Preservation of biologically active and other substances



2,34002 PRESERVATION oF BIOLOGICALLY ACTIVE AND OTHER smssTANcEsy E C.H. BARR Jan. 25, '1944.

Original Filed July l0, 1936 3 Sheets-Sheet 1 infn.

. Mk1/wrm? cadena/v0 Af. BAR/ BY Jan. 25, 1944. c. H. BARR PRESERVATIONOF; BIOLOGICALLY ACTIVE AND OTHER SUBSTANQEYS.

s sheets-sheet Original Filed July 10. 1936 Till 5.?. l Vall-.-

Aff-n C. H. BARR PRESERVATION OF BIOLOGICALLY ACTIVE AND OTHERSUBSTANCES Original Filed July l0, 1936 3 Sheets-Sheet 5 caf/mz M0 BARR@y Q Q @a fmf of vthe material Patented Jan. 25, 1944 UNITEDA sTATEsPATENT oFFlcE PRESERVATION F BIOLOGICALLY ACTIVE AND OTHER SUBSTANGESCourtland H. Barr, Glenolden, Pa.,

assignor to Sharp & Dahme, Incorporated, Philadelphia, Pa., acorporation of Maryland original application July-io, 193s, serial Na If 90,033. Divided and this application July 7,

1939, Serial No. 283.284

s claims. (ol. 12a-'272) The present invention relates to improvementsin the preservation of biologically active and improved methods andapparatus but also the improved nal containers containing suchdesiccated substances, sealed under the 'original vacuum, and

,provided with means by which water or other aqueous liquid can bereadily introduced into the container without destroying the vacuum, torestore the desiccated substances to a liquid state suitable for use.

Desiccated `biologically active substances are very porous, and it isdesirable to package and distribute them undei'. avacuum, both toprevent any deterioration which might be caused by' contact with theair, and to facilitate the dissolving in water or other aqueous uid whenrestoration is desired. If such material is 'maintained under a vacuum,and wateris introduced into the container before the vacuum is broken,the vacuum tends to pull the water into the poresA and interstices ofthe material, insuring intimate contact of the water with the materialand preventing the material from being impregnated with gas which mighttend to prevent the ready access of Water to the interior portions ofthe desiccated material. Methods of producing such desiccatedbiologically active substances, in

iinal containers, sealed under a high vacuum in containers provided withall-glass seals, and perforable closures sealed within the glass havebeen developed by Dr. Reichel of the Mulford Laboratories. Suchcontainers enable the distribution and storage of the materials whilemaintained under a high vacuum, With absolute assurance thai-.the vacuumis maintained and that the material is kept free 'from contamination,and yet permit water to be introduced without breaking the yacuum, bybreaking the glass seal over the periorable closure, and introducingwater by means of a hollow needle through this closure, which serves tohold the vacuum after the glass seal is broken.

It is an object of the present invention to provide an improved processand apparatus by which desiccated biologically active and othersubstances may be produced and stored in a container sealed with anall-glass seal and with a perforable stopper, such as a rubber stopper,sealed within the glass; and to provide such a container, having a bodyportion and a single neck, with a perforable stopper sealed within theneck, the material being sealed within the container under the originalvacuum, such that there l is absolute assurance that the vacuum ismaintained during storage, etc., and that the material is kept free fromcontamination, air, moisture, etc., and such that, when it is desired torestore the material to a liquid state, water or other liquid .may'beintroduced without destroying the vac- In accordance with the presentinvention, a suitable vial or container of ordinary soft glass,preferably cylindrical ih shape, provided with a relatively long tubularneck, the inside of which at the point adjacent the top of the vial orcon tainer is tapered to lit a. rubber stopper', is pro` vided. The sizeof the vial may be varied almost at willl its capacity being determinedby the amount ol material which it is intended to contain and being suchas to contain one or moreL unit portions of the material. Thus thecapacityl of the container may vary from a fraction of la cubiccentimeter to 50 or l00 or more cubic cen-v timeters.

Where the container is intended for the storage and'distribution oi'deslccated biologically active substances, which are to be desiccated inthe contalner, its volume should be somewhat more than' twice as greatas the volume of the material in# tended to be processed therein, inorder to pro-A.v

tion to its noi-mal liquid condition about fills the' container. Theneck of the vial, even where ta-v pered, must be of suiiicife'nt size topermit the free ow of water vapor during the deslccation process.

In carrying out the desiccation, the material is originally frozen byexposure to a refrigerant maintained at a very low temperature, and isthen subjected to the' action of a high vacuum, the heat absorbed duringthe sublimation or vaporization which takes place being suicient to'kmany constrictions.

Into the container so provided is introduced the proper amount ofbiologically active substance to be treated, and this substance is thenfrozen, preferably while the container is in a more or less horizontalposition to insure a. maximum exposed surface foi` sublimation, and byexposure to a refrigerant maintained at a very low temperature, e. g.--'70l C. The container is then attached to a vacuum manifold andsubjected to the action of a high vacuum to remove the water from thefrozen material. After the desiccation has proceededto the desiredextent, a rubber stopper is introduced into the neck of the vial andforced down to form a tight joint in the tapered portion of the neck ofthe vial or container. This is done without breaking the vacuum, and asa result, the material is sealed within the container under a highVacuum with ya preferable closure.

The glass neck of the vial, which extends considerably above the rubberstopper, is then flamesealed, either while the vial is still attached tothe vacuum manifold, or after removing the vial from the manifold anddrawing a portion of the neck to a capillary tube, re-evacuating theneck and name-sealing the neck at the point where it has been drawn to ane tube. It is advantageous to draw a portion of the necii. at the pointof sealing to a nne tube before sealing as this simplies the sealingoperation, and insures the production of a vacuum tight seal, withoutrisk of the glass puncturing, as frequently occurs when tubes ofrelatively large diameter, made from soft glass, are llame-sealed undera vacuum. After the sealing, or before, the tube or neck of the vial isetched or scratched a little below the top of the rubber stopper, sothat it may be readily broken oifat this point. The desiccated materialso produced is maintained as a formed porous mass, having the shape andvolume of the frozen material from which it is produced, without changein its physical structure after desiccation, and having an immensenetwork of capillaries or pores.

When it is desired to use the material within the container, it ismerely necessary to break off the neck or tube at the etched orscratched point and introduce water `or aqueous uid into the containerby means of a hypodermic needle or similar device which is readilyforced through the perforable vrubber stopper. The vacuum, which ismaintained within the container, during storage by means of the glassseal, and just prior t0 restoration by means of the rubber stopper,facilitates the introduction of water and insures the complete andspeedy dissolving of the material in the Water.

To carry out this process, and produce the final container containingthe treated material, the invention provides apparatus of suitableconstruction. In general, the apparatus comprises an attachment orapparatus suitably connected to a vacuum manifold, and provided withmeans for attaching containers, containing frozen material to bedesiccated, to it. The attachment also provides a passageway ofrelatively large diameter to permit the free flow of vapors from thecontalnerto the vacuum manifold. The attachment also includes means bywhich rubber Stoppers may be readily introduced into the neck of thevial after the desiccation is complete, and means by which the stoppersmay be forced into proper position to insure the production of a tightseal and the maintenance of a. high vacuum within the container eventhough the neck ofthe container is open and exposed to atmosphericpressure for relatively short periods of time, as `in name-sealing or inrestoring the desiccated material to a. liquid state.

The invention will be further illustrated and explained in connectionwith the drawings, which illustrate an attachment or apparatus whichembodies the invention and which illustrate a con- 4 tainer suitable formarketing and distribution containing a desiccated biologically activesubstance which embodies the invention, and which illustrate the meansby which water may be introduced into such container to restore thematerial to a liquid state without breaking the vacuum maintained withinthe container.

Inthe' drawings,

Fig. 1 is a sectional view of the attachment or apparatus shown in theposition used during the desiccation of the frozen material;

Fig. 2 is a sectional view of a portion of the apparatus or attachmentshowing the means by which a stopper is introduced into the neck of thevial or container;

Fig. 3 is a sectional view of a portion of the apparatus or attachmentshowing the means by which the rubber stopper is forced into place toform a tight seal;

Figs. 4 and 5 are views showing the final container before and after theglass seal is made;

Figs. 6, 7, and 8 are views showing the container after the glass sealis broken off and when the container is ready for the introduction ofwater and means by which water may be introduced;

Fig. 9 is a view in perspective, showing the attachment fastened to, avacuum manifold; and

Fig. 10 is a view of a portion of a suitable vacuum manifold, showing asuitable arrangement of outlets and means for fastening the attachmentsthereto.

In Fig. 1, l' represents a vial or container of suitable size intendedfor the marketing or distribution of a desiccated serum or otherbiologically active substance, containing a frozen or desiccatedbiologically active substance la, and provided with a relatively longneck 2 of suitable diameter and with a tapered portion 3 at the top ofthe vial or container. f This vial or container is connected to a metaltube 4 by means of a suitable vacuum-tight connection, as shown at 5.This connection, which has sucient tolerance to provide for variationsin the size of the container necks, includes a soft rubber tube 6 overwhich is slipped a, threaded brass cap 8. A portion of the rubber tubeis then turned back on itself as shown at 1, and a brass cap 9 screwedon to the threaded sleeve. The portion of the rubber tube under the capis thus of double thickness, and the cap limits the expansion of thisportion of the tube. When the neck of a container is inserted in therubber tube, and the vacuum applied, a vacuum-tight seal is quicklyformed. The metal tube 4 is provided with a side outlet i0 extendingupwardly at an angle.

' to tube 4 by means of an spiral or metal spring ings I Ia, throughwhich the vapors or gases from the container I flow tothe vacuummanifold. This hollow plunger is rigidly connected to metal tube I2, asat I2a. The tube I2 is also connected extensible tube I3, which consistsof an inner I4, and anouter or surrounding rubber tube I5, land which isreadily lengthened or shortened. stop I 4a, which may have a number ofholes IIb, is provided at the bottom ofthe tube I2 to conne the springI4.

The plunger II is of sufficient length to normally block the openingbetween the side outlet I and sleeve 4. The metal spring or spiral isprovided to prevent the rubber tube, which is reflativeLv long and mustbe extensible, from collapsing under the influence of the vacuum. Theconnections between tubes 4 and I2 and tube are, ofcourse, vacuum-tight.Tube I2 'is connected to the vacuum pump or vacuum manifold by metaltube I6 and the rubber tube I1, which is made of relatively heavyrubber, andwhich may be clamped to permit the sealing off of the lowerpart of the apparatus from the vacuum pump or manifold as whendisconnecting, the vial or container from the attachment, etc.Attachedto the side outlet I0 by means of therubber-connection I8, is ametal tube I9, provided with a special valve 20. The rubber connectionI8 is used so that the tube I9: and otherl portions of the apparatusattached-thereto may be readily sealed oif from the rest of theattachment or apparatus by a vacuum-tight seal. Valve 20, which isincluded in'tube I9, consists` of a rotatable metal disc having anopening 2| adapted to receive a rubber stopper 'suitablefor sealing the.container or vial from the upper-.portion of tube I3., and to turnitaround and allow it to drop into tube I0. This valve may be provided`with a. Small air hole .22 to prevent the rubber stopper from adheringto the valve when it is turned. Connected to the tube I 9 by means-cf4rubber tube 23 is a tube 24, of glass or Vother material, adapted tohold a number of the rubber Stoppers, each upside down. The tube 24 isprovided at the top with a stopper 25 having a small air hole 2i, and anair filter made of absorbent cotton or similar material 2l. Thus when a.stopper is removed from this tube by means of the valve 20, this airfilter and air hole allow' suicient sterile air to enter the tube toprevent the formation of a vacuum therein which might interfere withfeeding the Stoppers through the valve.

This tube for the stoppers is supportedby means.

of a ring or similar device 28, which is of a construction which willpermit the tube to be takenl oi or replaced at will, and which willpermit the tube to move vertically with the lower portion of theattachment and with respect to the Upper portion.

An advantageous method of fastening the. attachments to a`vacuummanifold, and a Suitable manifold, are illustrated in Figs. 9 and 1Q. InFig. 9 is shown an attachment withl a container I attached to it, as inFig. 1, with a bell 40 over the connection and the neck of thecontainer.

'I'his bell keeps dust, etc., which-falls downwardly from coming intocontact with the connection 5; and helps to preserve sterility in theoperation. The attachment is provided with a tube 24 for the rubberstoppers, and with a somewhat modified form of air filter 4I on thistube. This air filter includes a glass tube 42, containing absorbentcotton or" the like 42 attached to tube 24 by rubber connection 44. Theextensible tube I3 is fastened to a metal tube I2, as in Fig. l.

' and this tube is provided with a threaded stud or bolt 45 carrying a'wing nut 46. 41 'is fastened to the other end of a vacuum-tightconnection. f

This rubber tube fits over an outlet 48 on vac uuxn manifold 49, whichmay be provided with Ille or more such outlets (Figs. 9 and 10). Eachoutlet 48 is provided with a forked support 50, the slot of which fitsthe attachment then the wing nut 46. In is rigidly supported by by meansof tube 4l, vacuum-tight connection. The outlets on the vacuum manifoldproject upwardly at an angle, so that the gases and vapors, which passup wardly through the tube I3, then pass downwardly through the tube 41into the outlet 4I and into the vacuum manifold, thus preventing thecontamination of thematerial in the contube I2 with being secured inplace by this way, the attachment tainer by the contents of the manifoldor its outV and on the tube between the stopper receptacle and the tube4. The wrapping around the rubber tube at the top of the apparatus isremoved, and the attachment fastened to the vacuum manifold, suitableprecautions being taken to insure sterility. The rest of the wrapping isthen removed, and, attachment, as in Fig. 9, the open is immersed inantiseptic solution.

A container I having suitably frozen material Iav within it is attachedas shown in Figs. 1 and. 9, with sterile technique, the clamp on theconnection to the vacuum manifold is removed and the vacuum -is appliedand the material desiccated or dehydrated. After the desiccation ordehydraend of the bell tion is complete, the clamp is removed from thelY maintaining the vacuum tube I8, a stopper 29is introduced into tubeIll by means of th'e valve, and the clamp is replaced on the tube I 8.The lower portion ofthe apparatus, including the container, is thenpulled down to the position shown in Fig. 2, the exten sible or iiexibletube I3 permitting this. When the lower portion of the lapparatus ispulled down, the tube AI I, being rigidly connected to tube I2, no 4 andlonger blocks the opening between Ill, and permits sleeves the rubberstopper 29 to fall into position in the neck of the vial or con-Ytainer. As soon as the rubber stopper is in the proper position in theneck of the container, the lower portion of theattachment orapparatus'is raised, as shown in Fig. 3, so that the plunger Il extendsdown into the neck of vial or container and forces the rubber stopperinto tight engage-f ment with the tapered portion of the neck of thevial, thus insuring a tight seal which will retain the high vacuum inperiods of time as are required during the sealing of the glass neck ofthe vial and during the res'- toration of the material to a liquidstate.

After the rubber stopper has been forced into tight engagement with theneck of the vial while moved, permitting air to enter the attachmentthrough the air filter. In this way, the vacuum in the attachment isbroken without the inside of the attachment being contaminated. The

A lrubber tube v over the bolt or stud 45,

the vacuum manifold, and, i is connected thereto by a.

if a bell is provided at the lower end of the the vial for such shortveach time a container neck as shown in Fig. 6,

sealed before the container sealed vial, as in Fig. 4, is then removed,and

the bell again immersed in antiseptic solution.

This series of operations may then be repeated several times, and anumber of containers may be so processed, with sterile technique, andwithout requiring that the attachment be sterilized is processed.

After the container is removed, a portion of the neck is heated by meansof a broad flame and drawn to capillary dimensions and the neck againconnected to a vacuum pump or manifold and sealed by means of a flame. Afinal container such as shown in Fig. -with an all-glass seal is thusobtained. The neck of the vial is scratched or etched as shown at30 inFig. 5 to simplify breaking off the upper portion of the whenrestoration is desired. The neck of the vial or container may be isremoved from the apparatus, but preferably the sealing is accomplishedafterward, as this simplifies the production of a proper seal, andavoids the difficulty encountered in sealing a relatively large tubemade of ordinary soft glass under a highv vacuum.

I have described the flame-sealing of the neck of the vial or containerwhile the neck is connected to a vacuum, to insure the presence of avacuum in the sealed neck between the rubber stQpper and the glass seal,as this is a particularly advantageous method of sealing thecontainers,'and is the method which I prefer to use. The container soproduced, having -a vacuum both in the `container proper, and a vacuumin the neck between the rubber stopper and the glass seal is the form ofcontainer which I prefer and which is particularly advantageous.Nevertheless, my invention is not limited to the production of suchcontainers, or to such containers, as the glass seal may be made withoutconnecting the neck to a vacuum, and without producing a vacuum withinthe neck, particularly with those containers in which the volume ofthecontainer is relatively large, and is many times as great as the volumeof the neck, or that portion of the neck between tife rubber stopper andthe glass seal, without departing from the scope of my invention. Whenthe neck is flame-sealed without first producing a vacuum within theneck, the air within is greatadmit air into the container so that thedissolved material can be removed, the needle of the hypodermic syringeis again inserted through the rub- 'ber stopper and the container andsyringe turned upside down, as shown in Fig. 8 and the liquid materialsucked into the hypodermic syringe, whereupon it is ready for injection.

During such times as no container is attached to the apparatus, asuitable clamp is placed on tube i1 toform a vacuum-tight connection toprevent breaking the vacuum within the vacuum manifold. A clamp iscustomarily used on rubber tube i8 to insure a vacuum-tight connectionto prevent'the leakage of air through stopper 23 and past valve 20 intotubes lo and 4. This clamp is taken of! to permit stoppers to passfromthe valve 20 down into the tube I0 at such times as are desired, andduring such times, the valve 20 is sufficiently tight to prevent theingress of much air, and-'if the attachment is used in conjunction witha manifold of suitable size, the amount of air which enters' at thetimeof introduction of a stopper intol the tube I0 is so small that there isalmost no noticeable change in the pressure within the apparatus.

clamp may also be removed after a 'rubber stopper has been inserted in acontainer, and when it is d e'sired to remove the container from theattachment, to permit breaking thevacuum in the container with sterileair suitably filtered.

1- When this is done, care should be taken that the iy attenuated by theheat required to seal the appreciable amount but little air presentglass and is free from any of moisture so that there is above the rubberstopper stopper into the container, and almost no moisture, so that,despite the fact that some air may enter the container, the amount whichcan enter is very minute, so that thevacuum within the container,particularly where the container is relatively large, is not impaired-to any great degree, and sufficient air to interfere with the propersolution of the material on the introduction of sage 33 (Fig. 3)extending part way through them After the to facilitate the passage ofthe needle. proper amount of water has been forced into the containerand the material Within the container is dissolved. the needle isremoved temporarily to to penetrate past the.

tion or by baking, and connected the the material is fairly `rubbertubeconnecting the attachmentto the vacuum manifold is clamped.. However,the clamp should not be left off of the rubber tube i8 for any longerperiod of time than is necessary.

It-,will be understood that the apparatus as described above is by wayof illustration, and that many modifications and embodiments of theinvention may occur to those skilled in the art which are within thescope of the present invention. For example, instead of providing a tubeor cylinder containing which may be used for the sealing of a number ofcontainers, a suitable tube containing but one stopper, and hence notrequiring a mechanism such as the valve 20 to introduce the Stoppersinto the tube Il) singly, may be used. The provision of a tubecontaining a number of Stoppers is particularly advantageous, because itpermits the continuous operation of the apparatus with a minimum oflabor and a minimum of changing of connections, etc., and simplifies theintroduction of a sterile stopper into each container. For example, atube equipped with a suitable air filter may be supplied with some 7 or8 or more stoppers, the entire tube and Stoppers being ster ilized in asuitable manner, as by steam sterilizato the apparatus by tube 23 withsterile precautions. With pers is assured, anda number of containersequal to the number of Stoppers contained in the tube may be processedwithout the requirement of sterilizing a stopper and container thereforeach time a container is processed.

In carrying out a desiccation process such as described, the periodrequired to desiccate prolonged, and it is desirable to conductA theoperation so that for each attachment one container may be processed perday. In order to. accomplishV this, it is necessary to have the actualprocessing time not more than 22 hours, leaving some 2 hours for sealingthe containers and replacing them with a number of Stoppers i air filter21 provided, the sterility of the stopand the sealing of the containers.

new containers. with frozen material in them.

The process and apparatus of the present invention greatly speeds up thechanging of containers In order to seal a container according to theprocess of the present invention, it is merely necessary to introduce astopperinto the` neck of the container by allowing a. stopper to passfrom the valve 20 Q foi-able stopper within and in tight engagement withsaid neck, said container containing porous, relatively friable piecesof a desiccated biologically active substance of a size greaterfthanthat which would permit their introduction into the container throughthe neck thereof, said pieces having sides of a shape conforming to poritions of the inside contour of the container, said into the tube I0,pulling down the lower part of the apparatus so that the stopper fallsinto the proper position in the neck of the vial, and then to push upthe lower part of the apparatus so that the plunger Il forces thestopper into tight engagement with the neck of the vial. The containermay then be removed from the apparatus, and a new container placedthereon, ready for processing. It is not necessary to flame-seal theneck of thecontainer immediately, as during a short period of exposurethe seal obtained by means of the rubber stopper is suiiicient to holdthe high vacuum within the container and to prevent ingress of moistureor any contamination.

While I have described the invention with particular reference to itsapplication to the production and sealing of desiccated biologicallyactive substances in a final container, it is not limited thereto; as inits broader aspects it includesi the new processand apparatus as appliedto the introduction of a perforable, stopper into the neck of thecontainer while maintaining a vacuum therein, and to the production ofan evacuated container having an all-glass seal, and having a perforableclosure sealed within it, the stopper being introduced and* forced intoplace while a vacuum is maintained within the container.v

Nevertheless, the invention is of particular advantage for theproduction of desiccated biologically active substances in aV sealedcontainer provided with a glass seal, and with a perforable stoppersealed within it, such that the glass seal affords absolute assurancethat the vacuum within the container. is maintained and that the ingressof moisture or contaminating substances is prevented, and yet which isadapted for the introduction of water or liquid through the perforablestopper, after the glass seal is broken, without breaking the vacuum.The invention includes this new container, containing a desiccatedbiologically active substance, having but a single -neck or seal andsealed under its original vacuum. In such containers, the perforablestopper is protected from contact with air, etc., and fromdeterioration, by being sealed within lthe glass.

This application is directed to the completed article obtained by theuse ofthe methodand the operation described in and is a division of mycopending application Serial No. 90,033, filed July 10, 1936, whichlatter covers the method and apparatus for preparing the article..

I claim: A-

1. An article of manufacture comprising an biologically active substancehaving been desiccated from the frozen state within the container andthe container having been closed underv the original 'vacuum used whiledesiccating the biologically active substance, said perforable stopperforming a portion of the envelope of that part of the glass containerenclosing the desiccated biologically active substance, said outerextension being sealed Vat its outer end to enclose the stoppertherewithin.

2. An article of manufacture comprising an evacuated container, saidcontainer having a neck and an outer extension therefrom. andaperforable stopper within and in tight engagement with said neck, saidvcontainer containing porous, relatively friable pieces of a desiccatedbiologically active substance of a size greater than that which wouldpermit their introduction into the container through the neck thereof,said pieces having sides of a shape conforming to portions of vtheinside contour of the container, said biologically active substancehaving been desiccated from the frozen state within the container andthe container having Ibeen closed under the original vacuum used whiledesiccating the biologically active substance, said perforable stopperforming a portion of the envelope of that part of the glasscontainerenclosing the desiccated biologically active substance, said outerYextension being sealed at its outer end to enclose the stoppertherewithin, the space within said I tively friable pieces .of adesiccatedbiologically evacuated container, said container having a neckInd Bn 011W! extension therefrom, md' De!" active substance of a sizegreater than that which' would permit their introduction into thecontainer through the neck thereof, said pieces having sides of a shapeconforming to portions of the inside contour of the container, saidbiologically active substance havingbeen desiccated from the frozenstate within' the container and the container having been closed underthe original vacuum used while desiccating the biologically activesubstance, said perforable stopper forming a portion of the envelope ofthat part of the glass container enclosing the desiccated biologicallyactive substance, said outer extension being sealed at its outer end toenclose the stopper therewithin.

COUR'I'LAND H. BARR.

